High affinity integrin polypeptides and uses thereof

ABSTRACT

Polypeptides comprising all or part of a variant integrin α subunit A domain or a variant integrin β subunit A-like domain are described. In solution or in membrane-associated form, the A domain or the A-like domain of the polypeptides of the invention exists predominantly in a high affinity conformation. In the polypeptides of the invention, referred to as variant integrin polypeptides, a crucial isoleucine residue (described in greater detail below) is absent. The isoleucine can be either deleted or replaced with different amino acids residue, preferably a smaller or less hydrophobic amino acid residue, e.g., alanine or glycine. Because the variant integrin polypeptides of the invention exist in solution or in membrane-associated form predominantly in a high affinity conformation, they are useful in screening assays for the identification of molecules that bind to (and/or mediate the activity of) an integrin. They are also useful for generating antibodies, e.g., monoclonal antibodies, which bind to the high affinity form of an integrin. Some such antibodies recognize an epitope that is either not present or not accessible on an integrin that is in a lower affinity conformation. The variant integrin polypeptides of the invention can be derived from any integrin α subunit or any integrin β subunit and could be used therapeutically. The variant integrin polypeptides preferably include a ligand-binding portion of an A-domain or an A-like domain.

RELATED APPLICATION INFORMATION

This application claims priority from provisional application Ser. No.60/221,950, filed Jul. 31, 2000, the contents of which is herebyincorporated by reference.

BACKGROUND

Integrins are heterodimeric receptors that mediate a wide variety ofimportant interactions both between cells and between cells and theextracellular matrix via ligand binding. All integrins have an α subunitand a β subunit. Within the α subunit a region referred to as the Adomain (or I domain) is known to be an important mediator of ligandbinding. A similar region, the A-like domain, is present in many βsubunits. Many integrins are thought to exist in two conformations, alow affinity state (the “closed” or “unliganded” conformation”) and ahigh affinity state (the “open” or “liganded” conformation), the laterof which is responsible for high affinity ligand binding.

Integrins transduce signals that mediate the effects of the matrix onthe physiological activity of cells (e.g., motility, proliferation,differentiation). Moreover, integrins play a role in inflammation and inoncogenic cell transformation, metastasis, and apoptosis. Thus, there isconsiderable interest in identifying compounds that can activate orinhibit the activity of one or more integrins.

In order for an efficient integrin-ligand binding to occur, it isthought that the integrin must be in its high affinity configuration. Itappears that inside-out signals generated when cells are activated by avariety of stimuli apparently switch integrins from a low affinity stateto a high affinity state. This functional upregulation is associatedwith conformational changes in the extracellular regions of integrinsthat include the A domain of the α subunit and the A-like domain of theβ subunit (Smith et al. 1988 J. Biol. Chem. 263:18726).

The integrin A-domain assumes a dinocleotide-binding fold (Lee et al.1995 Cell 80:631; Emsley et al. 1997 J. Biol. Chem. 272:28512; Li et al.1998 J Cell Biol. 143:1523; Nolte et al. 1999 FEBS Lett. 452:379; andLegge et al. 2000 J. Mol. Biol. 295:1251), with a metal ion dependentadhesion site (MIDAS) on its top, and is connected through a C-terminalα7 helix at its bottom to the body of the integrin. MIDAS and itssurrounding exposed side-chains form the binding site for physiologicligands (Li et al., supra; Michishita et al., Cell 72:857–867, 1993;Kamata et al., J. Biol. Chem. 269:26006–26010, 1994; Kern et al., J.Biol. Chem. 269:22811–6, 1994; Edwards et al., J Biol Chem.273:28937–44, 1998; Zhang et al., Biochemistry 38:8064–71, 1999) andcertain antagonists (Rieu et al., J Biol Chem. 271:15858–15861, 1996).In the “open” conformation, three non-charged resides in the proteindirectly coordinate the metal ion in MIDAS, a pseudoligand or ligandglutamate residue (Lee et al., supra; Li et al., supra; Emsley et al.,Cell 100:47–56, 2000) completes metal coordination. In the “closed”form, the amphipathic C-terminal α 7 helix is shifted upwards by 10 Åcompared to the “open” form, wrapping around the rest of the domain.This large shift is associated with a change in metal coordination,where one of the three coordinating residues, a threonine, is nowreplaced with an aspartate, and a water molecule replaces the glutamatein completing the metal ion coordination sphere (Lee et al., Structure3:1333–1340, 1995). These changes in metal coordination and topology ofMIDAS are similar to those described in the structurally homologous Gproteins (Lee et al., supra).

The crystal structure of four integrin A-domains (CD11b, CD11a, CD49aand CD49b) have been reported to date (Lee et al., supra; Lee et al.,Structure 3:1333, 1995; Emsley et al., supra; Li et al., supra; Emsleyet al., J Biol Chem 272:28512, 1997). All with the exception of integrinCD11b A-domain (11bA), were found only in the “closed” form, leading tothe suggestion that the “open” form is a non-informative crystalartifact (Baldwin et al., Structure 6:923–935, 1998). Three studiessupport the view that the “open” form of the integrin A-domain equateswith the “high” affinity state (Li et al. 1998 J. Cell Biol 143:1523;Rieu et al., supra; Oxvig et al., Proc Nat'. Acad Sci USA 96:2215–20,1999). In the first, point mutations in CD11bA that are predicted onstructural grounds to destabilize the “closed” structure, increased theproportion of the “high affinity” form in solution (Li et al. 1998 J.Cell Biol 143:1523). In the second, the binding site for an“activation-dependent” monoclonal antibody mapped to a conformationallysensitive region of the A-domain (Oxvig et al., supra). The third studyshowed that an A-domain in complex with a short collagen peptide assumedthe “open” conformation, and suggested that the “open” form can only beobtained in the presence of ligand (Emsley et al., supra). While it hasbeen suggested that the ligand causes the conformational change inintegrins, at least one study suggests that integrins can exist in highaffinity state even in the absence of ligand (Smith and Cheresh 1988 JBiol Chem 263:18726–31). In addition, several studies suggested thatligand binding affinity in heterodimeric integrins can be altered in anallosteric manner (Li et al. 1998 J Cell Biol 143:1523; Edwards et al, JBiol Chem 273:28937–28944, 1998; Calderwood et al, J Biol Chem 273:5625,1998; Zhang et al., J Biol Chem. 271: 29953–7, 1996).

SUMMARY

The invention features polypeptides comprising all or part of a variantintegrin α subunit A domain or a variant integrin β subunit A-likedomain. In the polypeptides of the invention, referred to as variantintegrin polypeptides, an important isoleucine residue (described ingreater detail below) is absent. The isoleucine can be either deleted orreplaced with different amino acids residue, preferably a smaller orless hydrophobic amino acid residue, e.g., alanine or glycine. Becausethe variant integrin polypeptides of the invention tend to exist insolution in a high affinity conformation, they are useful in screeningassays for the identification of molecules that bind to (and/or modulatethe activity of) an integrin. They are also useful for generatingantibodies, e.g., monoclonal antibodies, which bind to the high affinityform of an integrin. Some such antibodies recognize an epitope that iseither not present or not accessible on an integrin that is in a loweraffinity conformation. Thus, the invention features antibodies whichbind with greater affinity to a variant integerin of the invention thanto corresponding wild-type integrin. The variant integrin polypeptidesof the invention can be derived from any integrin α subunit or anyintegrin β subunit. The variant integrin polypeptides preferably includea ligand-binding portion of an A-domain or an A-like domain.

The invention also features methods for identifying a compound thatbinds to a variant integrin polypeptide of the invention. Such screeningmethods can entail exposing the polypeptide to a test compound ofinterest and determining whether the compound binds to the polypeptide.Thus, the assay can be a simple binding assay (e.g., where binding ofthe compound is measured only in the absence of the ligand) or acompetitive binding assay (e.g., where binding of the compound ismeasured in the presence of the ligand). The invention also featuresmethods for identifying a compound that interferes with the binding ofan integrin ligand to an integrin by measuring the binding of anintegrin ligand to a variant integrin polypeptide in the presence andabsence of a test compound. The ability of a test compound to interferewith the binding of an integrin ligand to an integrin may also betested. In addition, the binding specificity of a compound can beassessed by measuring the binding of the integrin ligand to a secondintegrin (or measuring the binding of a second ligand to the integrin)in the presence and absence of the test compound.

The invention also features methods for interfering with the binding ofan integrin to an integrin ligand by administering a variant integrinpolypeptide of the invention or an antibody that selectively binds to avariant integrin polypeptide of the invention.

The invention also features methods of administering a variant integrinpolypeptide of the invention or an antibody that selectively binds to avariant integrin polypeptide of the invention for the purpose ofidentifying the presence of a ligand which binds to an active integrrin.Such assays are useful for diagnosing inflammation, e.g., occultinflammation (e.g. abscess or an active atherosclerotic lesion).

The invention further features nucleic acid molecules (e.g., mRNA andDNA) encoding a polypeptide of the invention or a polypeptide whichincludes a polypeptide of the invention. The invention also includesnucleic acid molecule encoding a fusion polypeptide comprising apolypeptide of the invention and a second polypeptide, e.g., animmunoglobulin constant domain.

The experiments described below concern the design a preparation of avariant form of CD11b (an integrin α subunit) that is more active thanthe wild-type form of CD11b. Without being bound by any particulartheory, it appears that, in solution, the amount of this variant subunitthat is in the open (active) conformation is greater than for thecorresponding wild-type form of the subunit.

Creation of the variant CD11b involves deletion or substitution of aninvariable C-terminal Ile residue. Deletion or substitution of the Ileresidue confers a high affinity phenotype in isolated CD11b A domain aswell as in the intact integrin. Moreover, The Ile-modified A-domain canbe crystallized in the “open” conformation. Thus, without being bound byany particular theory, it appears that an Ile-based allosteric switchcontrols affinity and conformation in the integrin A-domain.Accordingly, variant, high affinity forms of other integrin α subunits(and β subunits) can be created by deleting or substituting thecorresponding highly conserved Ile residue.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B depict surface representation of the CD11bA domaincrystal structure in its “closed” (FIG. 1A) and “open” (FIG. 1B) states,with the C-terminal α7 helix outlined as a gray ribbon, the isoleucine(I316) residue in black. An arrow points to the MIDAS face. MIDAS andSILEN (socket for isoleucine) lie on almost opposite ends of theA-domain structure.

FIGS. 1C and 1D depict magnified face views of SILEN residues (I135,L164, I236, Y267, lying within 4 Å radius from I316) in the “closed”(FIG. 1C) and “open” (FIG. 1D) conformations. In the “closed” form (FIG.1C), I316 sidechain is wedged in SILEN; L312 is seen on top. L312 movesto cover SILEN in the “open” structure (FIG. 1D). FIGS. 1A–1D were builtusing GRASP (Barry Honig, Columbia University, NY).

FIGS. 1E and 1F depict stereo views of the I316 coordination socket inthe “closed” (FIG. 1E) and “open” (FIG. 1F) conformations. SILENresidues and I316 and L312 are labeled.

FIGS. 2A and 2B depict 2F₀-F_(c) electron density maps of the C-terminalportions of α7 from 11bA¹²³⁻³²¹ (FIG. 2A) and 11bA¹²³⁻³¹⁵ structures.The maps were contoured at 1.1σ, and made with O.

FIGS. 2C and 2D depict ribbon diagrams showing the completesuperimposition of the 11bA¹²³⁻³²¹ (FIG. 2C, red tracing) and11bA¹²³⁻³¹⁵ (FIG. 2D, red tracing) structures on the reported “closed”(FIG. 2C, green tracing) and “open” (FIG. 2D, green tracing) forms ofthe CD11b A domain, respectively. The metal ion is shown as a purplesphere.

FIGS. 2E and 2F depict stereo views of MIDAS in the 11bA¹²³⁻³²¹ (FIG.2E) and 11bA¹²³⁻³¹⁵ (FIG. 2F) structures. The direct D242-metal bond,and indirect T209-water-metal bond (FIG. 2E) are characteristic of the“closed” conformation. The meta lion in this case is Mn²⁺. The MIDASconformation (a direct T209-metal bond, indirect D242-water-metal bond,and a pseudo ligand glutamate, occupying the active site, shown ingreen, and directly coordinating the metal ion (Ca²⁺) are those of the“open” conformation). In FIGS. 2E and 2 g, the metal ion is shown inpurple, and hydrogen bonds as dotted yellow lines.

FIGS. 3A–3J depict the results of functional analysis of 11bA^(E-G) and11bA¹²³⁻³¹⁵ A-domains using BIAcore™ for recording the interactions of11bA^(E-G) (FIGS. 3A–3E) and 11bA¹²³⁻³¹⁵ (FIGS. 3F–3J) with theactivation-dependent ligands iC3b (FIGS. 3A and 3F), fibrinogen (FIGS.3B and 3G) or CD54 (FIGS. 3C and 3H). All three ligands bound to11bA¹²³⁻³¹⁵, but not to 11bA¹²³⁻³²¹. The observed differences in bindingwere not due to differences in protein concentration, as binding to theactivation-independent ligands, NIF (FIGS. 3D and 3I) and mAb 904 (FIGS.3E and 3J) were comparable. To quantitatively determine the affinity,various concentrations of the A domain peptides were used. The bindingdata were analyzed by linear transformation, giving dissociationconstants (Kd, mean±SD, n=2) of 0.46±0.15 μM (for iC3b), 0.25±0.07 μM(for fibrinogen), and 0.22±0.04 μM (for CD54).

FIG. 4A depicts the results of an analysis of the binding of 11bA^(I→G)to iC3b. The dotted line represents the lack of binding of 11bA¹²³⁻³²¹to the same ligand. The calculated Kd is 0.66±0.3 μM (mean±SD, n=2).

FIG. 4B depicts a histogram (mean±SD, n=3) showing the relative bindingof^(I→G) CR3 expressed on COS cells to iC3b, compared with wild-typereceptor.

FIG. 5 depicts an alignment of the A domains of nine alpha integrin αsubunit (CD11b (SEQ ID NO:1), CD11c (SEQ ID NO:2), CD11d (SEQ ID NO:3),CD11a (SEQ ID NO:4), alpha 1 (SEQ ID NO:5), alpha 2 (SEQ ID NO:6), alpha10 (SEQ ID NO:7), alpha 11 (SEQ ID NO:8), and alpha E (SEQ ID NO:9)). Inthis alignment, the invariant Ile (I316) is indicated by an arrow.

FIG. 6 shows that an I to G mutation of the invariant isoleucine inCD11a A domain induces an activated domain in this integrin,underscoring the applicability of the findings in the CD11b A domain toother integrin A-domains.

FIG. 7 is an alignment of the A-like domains of eight integrin βsubunits (b3 (SEQ ID NO:10), b5 (SEQ ID NO:11), b6 (SEQ ID NO:12), b1(SEQ ID NO:13), b2 (SEQ ID NO:14), b7 (SEQ ID NO:15), b8 (SEQ ID NO:16),and b4 (SEQ ID NO:17)). In this alignment, the residue corresponding tothe invariant Ile in β subunits is indicated by an arrow.

DETAILED DESCRIPTION EXAMPLE 1

Generation of a Stable, High Affinity CD11b Variant A-Domain by Deletion

A variant CD11b A domain consisting of amino acids E¹²³ to K³¹⁵ of CD11b(11bA¹²³⁻³¹⁵; also referred to as 11bA^(E-K)) was created. Note that theamino acid numbering in these example corresponds to the mature protein.While the amino acid numbering in Tables 2 nad 3 belwo referes to thenumbering in the complete protein (including signal sequence). CD11b hasa 16 amino acid signal sequence. Thus, E¹²³ in the mature proteincorresponds to E139 in the complete protein. This variant A domain wascharacterized and compared to a CD11b A domain consisting of amino acidsE¹²³ to G³²¹ (11bA¹²³⁻³²¹; also referred to as 11bA^(E-G)). A variant Adomain with a Ile to Gly change at residue 316 (11bA^(I→G)) was alsocreated. All three proteins were expressed as GST fusion proteins thatwere cleaved to release the protein of interest.

The variant polypeptides were created using standard recombinanttechniques. Restriction and modification enzymes were purchased from NewEngland Biolabs, Inc. (Beverly, Mass.), Boehringer Mannheim (Germany),or GIBCO BRL (Gaithersburd, Md). Site-directed mutagenesis was carriedout in pGEX-4T-1 vector as described (Rieu et al. 1996 J Biol Chem271:15858). The following mutagenic primers were used. IFAdel Fwd:5′-TATAGGATCCGAGGCCCTCCGAGGGAGTCCTCAAGAGGATAG-3′(SEQ ID NO:18); Reverse:5′-CTACTCGAGTTACTTCTCCCGAAGCTGGTTCTGAATGGTC-3′(SEQ ID NO:19); I–Greverse: 5′-CTACTCGAGTTAACCCTCGATCGCAAAGCCCTTCTC-3′(SEQ ID NO:20).Introduction of the respective mutation was confirmed by direct DNAsequencing. The PvuI-BspEI-restricted cDNA fragment of the A-domaincontaining the mutation was subcloned into the PvuI-BspEI-restrictedCD11b cDNA, cloned into pcDNA3 plasmid, which containing full-lengthhuman CD11b (Rieu et al. 1996 J Biol Chem 271:15858). 11bA¹²³⁻³²¹ and11bA¹²³⁻³¹⁵ and 11bA^(1→G) A-domains were expressed as GST fusionproteins in Eseherichia coli (Michishita et al. 1993 Cell 72:857),cleaved with thrombin and purified as described Li et al. 1999 J. CellBiol 143:1523. C¹²⁹ was replaced by S in all the expressed GST-A-domainfusion form to prevent formation of disulfide-linked dimmers in solutionafter thrombin cleavage (not shown). Purity was confirmed by SDS-PAGEanalysis.

The structures of 11bA¹²³⁻³²¹ and 11bA¹²³⁻³¹⁵ were determined by x-raycrystallography. Crystals were grown using 10 mg/ml stock proteinsolutions and the hanging drop vapor diffusion method as described Li etal. (1998 J. Cell Biol 143:1523). Several crystal conditions were triedfor each A-domain. 11bA¹²³⁻³¹⁵ and 11bA^(I→G) formed crystals in thepresence of a reservoir solution containing 15% polyethylene glycol 8 K,0.10 M Tris, pH 8.2, 150 mM CaCl₂, at room temperature. Crystals startedto form within a week, grew to a typical size of 0.3 mm×0.05 mm×0.04 mmin two weeks, and belonged to the tetragonal space group P4, with unitcell of a=b=45.7 Å. 11bA¹²³⁻³²¹ did not crystallize under theseconditions, but formed crystals at room temperature using 10%polyethylene glycol 4000, 0.1M sodium citrate, pH 4.5, 5 mM MnCl₂ in thereservoir buffer. 11bA¹²³⁻³²¹ was crystallized in the P2₁2₁2₁ spacegroup with unit cell of a=48.1 Å, b=121.5 Å, c=74.5 Å.

A single 11bA¹²³⁻³¹⁵ crystal was used to collect a 2.3 Å resolution dataset, at 100 K, on beamline X12B of the National Synchrotron Light Sourceat the Brookhaven National Laboratory using a CCD detector. A single11bA¹²³⁻³²¹ crystal was used to collect a 2.6Å resolution data set, at100K, using in-house X-ray on image plate. DATA were processed withDENZO and SCALEPACK to an Rsym of 8.8%, 7.7%, respectively. The startingmodels were the refined 1.8 Å Mg²⁺structure (pdb accession code lido)(Lee 1995 Cell 80:631) comprising residues D132 to K315, and the refined2.0 Å Mg²⁺-structure (pdb accession code 1jlm) (Lee et al. 1998Structure 6:923), comprising residues D132 to A318, with the metal andwater molecules removed, respectively. The preliminary rigid bodyrefinements were performed using X-plor, with the diffraction data rangefrom 8.0 to 3.0 Å resolution with 5% of reflections for R-freecalculation. The phases were gradually extended to high resolution andthe structures were refined by several cycles of alternatingtorsion-angle dynamics and restrained individual isotropic B factorrefinement protocols with all diffraction data between 8.0 and 2.6 Å,and 8.0 and 2.3 Å resolution, for 11bA¹²³⁻³²¹ and 11bA¹²³⁻³¹⁵structures, respectively (Table 1). Model inspection and manualadjustments were made on SGI graphics workstations using O (Jones et al.1991 Acta Crystallogr 47:110). The addition of solvent molecules wasbased on the suitable peaks in difference maps, reasonable hydrogen bondand refined temperature factors of less than 50 Å². The addition ofsolvent molecules was based on the suitable peaks in difference maps,reasonable hydrogen bond and refined temperature factors of less than 50Å². The structure was refined to a final R factor of 20.3% (R-free:25.6%) for the 11bA¹²³⁻³¹⁵ structure, and 21.9% (R-free: 30.0%) for the11bA¹²³⁻³²¹ structure. The final models comprises all nonhydrogen atomsof residues D132 to K315, 30 water molecules, and one Ca²⁺ ion for11bA¹²³⁻³¹⁵, and residues D132 to G321, 44 water molecules and one Mn²⁺ions for 11bA¹²³⁻³²¹. Crystallographic data is presented in Table 1.

TABLE 1 123–321 123–315 I316→G 11bA 11bA 11bA Space group P2₁2₁2 P4₃ P4₃Unit cell constants (Å) a = 48.1, b = 121.5, a = b = 45.7 a = b = 45.2,c = 74.6 c = 94.8 c = 95.0 No. of unique 13,990 7,955 3,749 reflectionsResolution (Å) 8.0–2.6 8.0–2.3 8.0–3.0 ^(R)merge (%)* 7.7 {23}^(§) 8.8{28}^(§) 11.2 {30.4}^(§) Completeness (%) 99.3 {98.4}^(§) 91.1{80.8}^(§) 95.3 {73.6}^(§) Redundancy  5.9  2.3  2.7 R-factor^(l) (%)21.9 18.8 20.8 R-free^(‡)(%) 30.0 24.8 28.8 Solvent molecules 82 62 20Metal ions 2 (Mn) 1 (Ca) 1 (Ca) Root mean square (rms) deviations fromideal values Bond lengths (Å) 0.007 0.007 0.006 Bond angle (°) 1.3 1.31.25 *R_(merge) = Σ|I-<I>|Σ I where I is the observed intensity and <I>is the average intensity from multiple observations of symmetry-relatedreflections. ^(§)Numbers in {} shows values for highest 0.1 Å resolutionbin. ^(l) R-factor = Σ|Fo − Fc|Σ Fo. ^(‡)R-free = Σ_(T) | Fo − Fc|Σ_(T)Fo, where T is a test set containing a randomly selected 5% of thereflections omitted from the refinement.

The crystal structure 11bA¹²³⁻³²¹ was that of the “closed” conformation(FIGS. 2A, 2C, and 2E). In contrast, 11bA¹²³⁻¹¹⁵ crystallized in the“open” form (FIGS. 2B, 2D, and 2F).

The ligand binding properties of 11bA¹²³⁻³²¹ and 11bA¹²³⁻³¹⁵ weredetermined using surface plasmon resonance as previously described (Liet al., supra) on a BIAcore (BIAcore AB, Uppsala, Sweden). IC3b,fibrinogen or CD54 each was covalently couples via primary amine groupsto the dextran matrix of a separate CM5 sensor chip (BIAcore AB). BSAimmobilized in the same way was used as a control surface. 11bA¹²³⁻³²¹,11bA¹²³⁻³¹⁵ and 11bA^(I→G) A-domains were flowed over the chip at 5ml/min at different times. TBS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl)with 2 mM MgCl₂ and 0.005% P20 (BIAcore AB) was used as the runningbuffer. 1 M NaCl in 20 mM Tris-HCl, pH 8.0, was used to remove the boundproteins and to regenerate the surface. Binding was measured as afunction of time. The binding data (after subtracting background bindingto BSA-coated chip) were analyzed using Scatchard plots as described(Dall'Acqua et al. 1996 Biochemstry 35:9667–9676). COS M7 simianfibroblastoid cells at ˜70% confluence were transfected with supercoiledcDNAs encoding WT or CD11b^(I□G) together with full-length CD 18 asdescribed (Michishita et al., supra)

Transfected COS cells were grown for 24 h in Iscove's modifiedDulbecco's medium (BioWhittaker, Inc., Walkersville, Md.) supplementedwith 10% FBS, 2 mM glutamine, 50 IU/ml penicillin and streptomycin at37° C. Cells were washed, detached with 0.1% trypsin-EDTA, and seeded inreplicates for 24 h onto 24- or 48-well plates (Costar Corp., Cambridge,Mass.) or 100-mm petri dishes. Confluent monolayers in 24- or 48-wellplates were used for cell-surface antigen quantification andligand-binding studies, and those on petri dishes forimmunoprecipitation studies. Heterodimer formation and binding ofiC3b-coated erythrocytes to wild type and CR3^(I→G) holoreceptors werecarried out as described (Rieu et al. 1996 J Biol Chem 271:15858).Specific binding of iC3b to the holoreceptors was obtained bysubtracting background binding to mock-transfected COS cells. Binding toCR3^(I→G) was expressed as a percentage of binding to wild type, aftercorrecting for the degree of surface expression using binding of mAb904(Rieu et al. 1996 J Biol Chem 271:15858).

As shown in FIGS. 3A–3J, 11bA¹²³⁻³²¹ showed no binding to theactivation-dependent “physiologic” ligands, complement iC3b, firbrinogenand CD54 (ICAM-1). In contrast, 11bA¹²³⁻³¹⁵ displayed high affinitybinding to all three ligands (FIGS. 3A–3J). Both domains bound equallywell to the activation-independent “ligands” NIF (neutrophil inhibitoryfactor) (FIGS. 3D, and 3I), and mAb 904 (FIGS. 3E and 3J), indicatingthat the differences observed are not caused by variations in A-domainconcentrations. These data conclusively establish that the “open” and“closed” crystal structures correspond respectively to the “high” and“low” affinity states of integrin 11bA.

EXAMPLE 2 Generation of a Stable, High Affinity State CD11b Variant bySubstitution

Ile³¹⁶ is invariable in all integrin alpha A-domains cloned to date(FIG. 5). An A-domain with an Ile to glycine substitution (11bA^(I→G))exhibited a “high affinity” state (FIG. 4A). The same substitutioncreated in the holoreceptor dramatically increased its ligand bindingactivity (FIG. 4B). The crystal structure 11bA^(I→G) was identical tothat of the “open” 11bA¹²³⁻³¹⁵ form. These data firmly establish thatthe “open” high affinity conformation is primarily dictated by anIle-based switch, intrinsic to the domain, and acting allosterically toregulate ligand binding affinity on the MIDAS face. It is known that theinactive an active conformers of an integrin exist in the absence ofligand (Smith et al., supra; Yan et al., J. Biol. Chem. 275:7249–60,2000). Based on the results described above, it appears that the role ofthe ligand is not in initiating the high affinity state as has beenrecently proposed (Emsely et al, supra), but in stabilizing it, throughshifting the equilibrium between the low and high affinity state infavor of the latter (Li et al., supra). Integrin activation byinside-out signaling would lead to a shift in this equilibrium,increasing the proportion of high affinity receptors on the cell surfacethat become “available” for ligand binding. Ligand engagement would thengenerate new epitopes, perhaps extrinsic to the A-domain, that initiateoutside-in signaling.

A conserved hydrophobic intramolecular socket (SILEN, Socket forIsoleucine), fastens the I³¹⁶ finger in the “closed” conformation; I³¹⁶is replaced by L³¹² in the “open” structure (FIGS. 1A and 1F). SILEN isformed by the hydrophobic side chains of I¹³⁵, L¹⁶⁴, I²³⁶, and Y²⁶⁷ bothin the “closed” and “open” conformations. (FIGS. 1C and 1D). In severalintegrins, certain mutations that lie outside MIDAS produce gain-offunction effects in the holoreceptor, and are believed to actallosterically (Zhang et al., supra; Oxvig et al., supra; Zhang et al.,supra). These studies were carried out in the holoreceptors, making itdifficult to provide a mechanistic interpretation, because of potentialinterdomain interactions and/or other quaternary effects. Thesemutations occur in or around SILEN. For example, substitution of theα1-βB loop of CD11b with that of CD11a, generates a constitutivelyactive integrin (Zhang et al., supra). This region spans one of theSILEN residues L¹⁶⁴. Integrin activation also occurs in an L¹⁶⁴-Fsubstitution (Oxvig et al, supra), which predictably makes SILEN smallerand therefore less accommodation to I³¹⁶. Other activating mutationsinvolving E¹³¹, D¹³², K²³¹ and F²³⁴ lie at the bottom of the structure,in close proximity to SILEN (Oxvig et al., supra), and may thus exerttheir effect through interference with the proper coordination of theIle “finger” in SILEN. The inhibitory effect of certain mAbs withepitopes on the opposite side of MIDAS (e.g., mAb 44a, the epitope ofwhich spans residues on the top of SILEN) may similarly be explainedthrough stabilization of the SILEN pocket.

The presence of the N-terminal extension facilitates A-domain switchinginto the less favored high-affinity state (Li et al, supra). Theunderlying structural basis for this effect is unknown, since none ofthe residues in the N-terminal extension are included in the derived 3-Dstructures. It has been observed however that residues within thisextension regulate ligand binding in A-domains. First, naturallyoccurring point mutations in this segment of the vWF A1 domain causegain-of-function phenotypes in patients with type IIB vWf disease(Matsushita et al., J. Biol. Chem 279:13406–14, 1995). This region alsocontains an activating mutation in CD11b A domain (Oxvig et al., supra).Third, synthetic peptide from the N-terminal extension inhibitedCD11a-dependent adhesion. Structural data from the CD49b A-domain alsoshow that three residues that extend beyond the α7 helix can pack into acrevice formed in part by residues in the N-terminal extension, bringingthe N and C termini into very close proximity (Emsley et al., supra).Flexibility of the C-terminal residues in α7 has also been observed inthe crystal and NMR structures of the CD11a A-domain. Taken together,these data suggest the N-terminal extension may offer an alternative byimperfect “competitive” surface for luring Ile away from SILEN, allowingsome molecules to exist in the “open” form. The present data suggestthat such a mechanism may operate in the holoreceptor, providing amechanistic basis for integrin activation by inside-out signals.

Variant Integrin Polypeptides

Given the sequence similarity among the A domains of integrin αsubunits, deletion of substitution of the Ile in a selected integrin αsubunit that corresponds to Ile³¹⁶ of CD11b should result in thecreation of a variant integrin α subunit that is more active (i.e., insolution has a greater proportion of ligand binding form polypeptides)that the wild-type form of the subunit. FIG. 5 is an alignment of theC-terminal α7 helix of the A domains of nine integrin α subunits (CD11b,CD11c, CD11d, CD11a, alpha 1, alpha 2, alpha 10, alpha 11, and alpha E).In this alignment, the invariant Ile corresponding to Ile³¹⁶ of CD11b inthe other integrin α subunits is outlined (arrow). Replacing theinvariant Ile with Ala or Gly or some other suitable amino acid shouldcreate a variant integrin polypeptide with increased activity. We haveshown this to be the case in the CD11a A-domain (FIG. 6). A variantCD11a A domain containing an I to G substitution displays binding to theactivation dependent ligand ICAM-1 in an ELISA assay. No binding wasobserved in the wild-type protein without this substitution.Alternatively, the portion of the integrin α subunit (or the A domain)that includes the invariant Ile and all amino acid residues C terminalto the invariant Ile can be deleted. Table 2 below lists the position ofthe invariant Ile in each of the integrin α subunits depicted in FIG. 5.

TABLE 2 Invariant Ile Gen Bank Position in Integrin α Subunit AccessionNo. whole integrin A domain Human CD11b RWHU1B Residue 332 C144–A334Human CD11c RWHU1C Residue 333 C145–A335 Human CD11d AAB38547 Residue332 C144–A334 Human CD11a AAC31672 Residue 331 C150–V333 Human Alpha 1P56199 Residue 331 C139–A333 (CD49a) Human Alpha 2 NP_002194 Residue 361C169–S363 (CD49b) Human Alpha 10 XP_002097 Residue 249 C57–G251 HumanAlpha 11 NP_036343 Residue 349 C159–S351 Human Alpha E A53213 Residue385 E196–S387The present invention features variant integrin α subunit polypeptidesin which the invariant Ile (listed in Table 2) is substituted by Gly,Ala or some other amino acid (e.g., Val). The polypeptide can includepart or all of the indicated A domain, e.g., 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200contiguous amino acids of the A domain that includes the position of theinvariant Ile. The invention also includes variant integrin α subunitsin which the Ile has been deleted. Also within the invention arepolypeptides comprising the entire A domain of an integrin α except forthe invariant Ile. For example, amino acids 144–132 of CD11b, aminoacids 145–332 of CD11c, amino acids 144–331 of CD11d, amino acids150–330 of CD11a, but does not include the remainder of the integrin αsubunit. The invention also features polypeptides comprising the Adomain of an integrin α subunit up to but not including the invariantIle and further lacking the 5 amino acids following the invariant Ile(e.g., amino acids 144–331, but not 332–336 of CD11b; amino acids145–332, but not 333–337 of CD11c; amino acids 144–331, but not 332–336of CD11d; amino acids 150–330, but not 331–335 of CD11a; amino acids139–330, but not 331–335 of human alpha 1; amino acids 169–360, but not361–335 of human alpha 2; amino acids 57–248, but not 249–253 of humanalpha 10; amino acids 159–348, but not 349–353 of human alpha 11; oramino acids 196–384, but not 385–389 of human alpha E).

Given the sequence similarity among the A-like domains of integrin βsubunits, deletion or substitution of the Ile in a selected integrin βsubunit that corresponds to Ile³¹⁶ of CD11b should result in thecreation of a variant integrin β subunit that is more active (i.e., insolution has a greater proportion of ligand binding form polypeptides)than the wild-type form of the subunit. FIG. 7 is an alignment of theA-like domains of integrin β subunits. Replacing the conserved Ile withAla or Gly or some other suitable amino acid should create a variantintegrin polypeptide with increased activity. Alternatively, the portionof the integrin β subunit (or the A-like domain of the subunit) thatincludes the conserved Ile and all amino acid residues C terminal to theinvariant Ile can be deleted. Table 3 below lists the position of theconserved Ile in each of the integrin β subunits depicted in FIG. 7.

TABLE 3 GenBank Conserved Integrin β Subunit Accession No. Ile PositionA-like domain Human β2(CD18) XP_009806 L363 Y125–S365 Human β1 NP_002202L378 Y141–S380 Human β3 I77349 I377 Y136–S379 Human β4 AAC51632 I366S128–S368 Human β5 A38308 I378 Y136–S380 Human β6 NP_000879 L371Y131–S373 Human β7 NP_000880 L389 Y150–S391 Human β8 NP_002205 L384Y145–S386Nucleic Acid Molecules Encoding Variant Integrin Polypeptides

The invention features isolated or purified nucleic acid molecules thatencodes a variant integrin polypeptide, e.g., a full length variantintegrin subunit or a fragment thereof (e.g., in which the invariant Ileis deleted or substituted), e.g., a biologically active variant integrinpolypeptide.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence encoding amino acids 123–315 of theCD11b α subunit (SEQ ID NO:1) or a portion thereof. The nucleic acidmolecules can include non-coding sequences or sequences encoding all ora portion of a protein other than an integrin.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence encoding amino acids 123–315 of the CD11b α subunit.

A nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:1. For example, such a nucleicacid molecule can include a fragment which encodes all or a portion(e.g., an immunogenic or biologically active portion) or an integrin Adomain or A-like domain.

Variant Integrin Polypeptides

The present invention also includes variant integrin polypeptides. Suchpolypeptides can be produced using recombinant DNA methods, by chemicalsynthesis or using other techniques. The polypeptides can be used asimmunogens or antigens generate antibodies which bind the active form ofan integrin A-domain or A-like domain. The polypeptide can bepost-translationally modified, e.g., glycosylated.

A variant integrin polypeptide can be part of a fusion protein whichincludes all or a portion of a second polypeptide that is not a variantintegrin polypeptide. This second polypeptide can be fused to theC-terminus or the N-terminus of the variant integrin polypeptide. All orpart of a third peptide may also be present. Thus, a variant integrinpolypeptide can be fused to, e.g., GST, an immunoglobulin constantregion, a heterologous signal sequence.

The variant integrin polypeptide fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. For example, the variant integrin polypeptides can beused to reduce skeletal muscle injury. Isolated CD11b A domain hasalready been found to reduce damage in an animal model of this type ofinjury. Briefly, purified recombinant rat CD11b A-domain wasadministered intravenously in a single dose (1 mg/kg) to seven groups ofLewis rats (5 per group), 30 minutes before inducing mechanicallyskeletal muscle injury. Equal numbers of rats were treated with afunction-blocking anti-CD11b/CD18 mAb (1 mg/kg). Quantitativehistological examination of the wounded area in controlled rats (treatedwith PBS), showed edema, myofiber disruption, necrosis and erythrocytesextravasation. Influx of neutrophils was detected 30 minutes post wound,followed by a second wave 3 hours later. There was also significanttissue necrosis outside the immediate wounded area (5 mm zone)associated with the presence of activated neutrophils. A-domain ormAb-treated rats showed a comparable and significant decrease in thenumber of infiltrated PMN (75±10%, n=35) and a protection of themuscular fibers outside the immediate zone of necrosis (80±8%, n=35).These data show that the A-domain can be an effective tissue-preservingagent in this model of muscular injury. Variant A domain should beequally effective.

The variant integrin polypeptide fusion proteins can be used to affectthe bioavailability of a variant integrin polypeptide ligand.

Variant integrin polypeptide fusion proteins may be usefultherapeutically for the treatment of disorders caused by, for example,ischemia-reperfusion injury (Stroke 30:134–9, 1999), immune complexes (JExp Med 186:1853–63, 1997), restenosis, and parasitic diseases (e.g.Ancylostoma spp; see J Cell Biol 127:2081–91, 1994).

Moreover, the variant integrin polypeptide-fusion proteins of theinvention can be used as immunogens to produce anti-variant integrinpolypeptide antibodies in a subject, to purify variant integrinpolypeptide ligands and in screening assays to identify molecules thatinhibit the interaction of variant integrin polypeptide with an variantintegrin polypeptide ligand.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). An variant integrinpolypeptide-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the variantintegrin polypeptide protein.

Variant integrin polypeptides can be produced using an expressionvectors (e.g., a plasmid vector or a viral vector). The vector can beautonomously replicating or integrated in to the host genomes. Theexpression vector can include at least one regulatory sequence (e.g.,promoter, enhancer, polyA site, or a cell- or tissue-specifictranscription factor binding site) that is operatively linked to thenucleic acid sequence to be expressed. The expression vector can bedesigned for expression in prokaryotic or eukaryotic cells, e.g., plantcell, insect cells, yeast cells, mammalian cells, and E coli. Dependingon the host cells used to express the variant integrin polypeptide, itmay be desirable to encode the polypeptide using codons optimized forthe host cell. In some cases it may be desirable to employ an expressionvector capable of directing tissue-specific expression.

The invention also features host cells or recombinant cells harboring anucleic acid molecule encoding a variant integrin polypeptide. Thenucleic acid molecule can be integrated into the host cells genome orpresent in an autonomously replicating vector. A host cell can be anyprokaryotic or eukaryotic cell, e.g., E. coli, an insect cell, yeast ora mammalian cell. The nucleic acid molecules can be introduced into thehost cells through transformation or transfection techniques.

A host cell of the invention can be used to produce (i.e., express) avariant integrin polypeptide by culturing the host cell under conditionssuch that the polypeptide is produced and then isolating the polypeptidefrom the cells or the culture medium.

Antibodies Recognizing Variant Integrin Polypeptides

The invention also features antibodies directed against a variantintegrin polypeptide. Such antibodies bind to the variant polypeptidewith greater affinity than they bind to the corresponding wild-typeintegrin polypeptide. The antibodies can be generated using standardmethods and can be screened by comparing the binding of the antibody tothe variant integrin polypeptide to binding of the antibody to thecorresponding wild-type polypeptide.

The term “antibody” refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include F(ab) and F(ab′)₂ fragments which can be generated bytreating the antibody with an enzyme such as pepsin. The antibody can bea polyclonal, monoclonal, recombinant, e.g., a chimeric or humanized,fully human, non-human, e.g., murine, or single chain antibody. In apreferred embodiment it has effector function. The antibody can becoupled to a toxin or imaging agent for use in diagnosis of occultinflammation (e.g. in abscess or active atherosclerotic plaques).

Chimeric, humanized, but most preferably, completely human antibodiesare desirable for applications which include repeated administration,e.g., therapeutic treatment (and some diagnostic applications) of humanpatients.

The anti-variant integrin polypeptide antibody can be a single chainantibody which can be optionally dimerized or multimerized to generatemultivalent antibodies. The antibody can be designed to have little orno ability to bind to an Fc receptor.

The antibodies of the invention can be used to detect or purify integrinsubunits that are in the active conformation. Thus, they can be used toevaluate the abundance and pattern of expression of an active form of anintegrin as part of a clinical testing procedure. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labeling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials (e.g., horseradish peroxidase, alkalinephosphatase, β-galactosidase, acetylcholinesterase, streptavidin/biotinand avidin/biotin, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin, luminol, luciferase, luciferin, aequorin,¹²⁵I, ¹³¹I, ³⁵S and ³H).

Screening Assays to Identify Compounds that Interact with a VariantIntegrin Polypeptide

The invention features methods for evaluating a compound for the abilityto bind to a variant integrin polypeptide or inhibit the ability of anintegrin ligand (e.g., a naturally-occurring integrin ligand) to bind toan variant integrin polypeptide. The methods can include contacting thecompound with the variant integrin polypeptide in the presence orabsence of a ligand and measuring the ability of the compound or theligand to bind to the variant polypeptide. The methods can be performedin vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybridinteraction trap assay. The method can be used to identifynaturally-occurring molecules which interact with an integrin. It canalso be used to find natural or synthetic inhibitors of the interactionbetween an integrin and an integrin ligand.

The compounds tested can be, e.g., proteins, peptides, peptidomimetics,peptoids, and small molecules. The test compounds can be obtained fromcombinatorial libraries including: biological libraries; peptoidlibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; “one-beadone-compound” libraries.

Libraries of compounds may be presented in solution (e.g., Houghten 1992Biotechniques 13:412–421), or on beads (Lam 1991 Nature 354:82–84),chips (Fodor 1993 Nature 364:555–556), bacteria (Ladner U.S. Pat. No.5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull etal. 1992 Proc Natl Acad Sci USA 89:1865–1869) or on phage (Scott andSmith 1990 Science 249:386–390); Devlin 1990 Science 249:404–406; Cwirlaet al. 1990 Proc. Natl. Acad. Sci. 87:6378–6382; Felici 1991 J. Mol.Biol. 222:301–310; Ladner, supra.).

The screening assay can be a cell-based assay in which a cell whichexpresses an variant integrin polypeptide protein or biologically activeportion thereof is contacted with a test compound, and the ability ofthe test compound to modulate variant integrin polypeptide activity isdetermined. Determining the ability of the test compound to modulatevariant integrin polypeptide activity can be accomplished by monitoring,for example, the ability to bind an integrin ligand.

The ability of the test compound to modulate variant integrinpolypeptide binding to a compound, e.g., an integrin ligand or to bindto variant integrin polypeptide can also be evaluated. This can beaccomplished, for example, by coupling the compound, e.g., thesubstrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to variant integrin polypeptide canbe determined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, a variant integrin polypeptide or the integrinligand can be coupled with a radioisotope or enzymatic label to monitorthe ability of a test compound to modulate variant integrin polypeptidebinding to an integrin ligand. For example, compounds can be labeledwith ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

In yet another embodiment, a cell-free assay is provided in which anvariant integrin polypeptide or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the variant integrin polypeptide or biologically active portionthereof is evaluated. Preferred biologically active portions of thevariant integrin polypeptide to be used in assays of the presentinvention include fragments that bind an integrin ligand.

Soluble and/or membrane-bound forms of isolated proteins (e.g., variantintegrin polypeptide proteins or biologically active portions thereof)can be used in the cell-free assays of the invention. Whenmembrane-bound forms of the protein are used, it may be desirable toutilize a solubilizing agent. Examples of such solubilizing agentsinclude non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the variant integrinpolypeptide protein to bind to a target molecule, e.g., an integrinligand, can be accomplished using real-time Biomolecular InteractionAnalysis (BIA) as described above (see, e.g., Sjolander and Urbaniczky(1991) Anal. Chem. 63:2338–2345 and Szabo et al. (1995) Curr. Opin.Struct. Biol. 5:699–705). “Surface plasmon resonance” or “BIA” detectsbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the mass at the binding surface(indicative of a binding event) result in alterations of the refractiveindex of light near the surface (the optical phenomenon of surfaceplasmon resonance (SPR)), resulting in a detectable signal which can beused as an indication of real-time reactions between biologicalmolecules.

In one embodiment, the polypeptide or the test compound is anchored ontoa solid phase. The polypeptide/test compound complexes anchored on thesolid phase can be detected at the end of the reaction. Preferably, thepolypeptide can be anchored onto a solid surface, and the test compound,(which is not anchored), can be labeled, either directly or indirectly,with a detectable labels.

It may be desirable to immobilize either a variant integrin polypeptide,an anti-variant integrin polypeptide antibody or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay.Binding of a test compound to a variant integrin polypeptide protein, orinteraction of an variant integrin polypeptide protein with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/variantintegrin polypeptide fusion proteins or glutathione-S-transferase/targetfusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtiter plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or variant integrin polypeptideprotein, and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of variant integrin polypeptide binding or activitydetermined using standard techniques.

Other techniques for immobilizing either a variant integrin polypeptideor a target molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated variant integrin polypeptide or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith variant integrin polypeptide or target molecules but which do notinterfere with binding of the variant integrin polypeptide to its targetmolecule. Such antibodies can be derivatized to the wells of the plate,and unbound target or variant integrin polypeptide protein trapped inthe wells by antibody conjugation. Methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thevariant integrin polypeptide or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the variant integrin polypeptide or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, includingchromatography, electrophoresis and immunoprecipitation.

In a preferred embodiment, the assay includes contacting the variantintegrin polypeptide or biologically active portion thereof with a knowncompound which binds variant integrin polypeptide to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with an variantintegrin polypeptide protein, wherein determining the ability of thetest compound to interact with an variant integrin polypeptide includesdetermining the ability of the test compound to preferentially bind tovariant integrin polypeptide or biologically active portion thereof, orto modulate the activity of a target molecule, as compared to the knowncompound.

To identify compounds that interfere with the interaction between avariant integrin polypeptide and an integrin ligand, a reaction mixturecontaining the variant integrin polypeptide and the ligand is incubated,under conditions and for a time sufficient, to allow the two products toform complex. In order to test an inhibitory agent, the reaction mixtureis provided in the presence and absence of the test compound. The testcompound can be initially included in the reaction mixture, or can beadded at a time subsequent to the addition of the target gene and itscellular or extracellular binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the variant integrin polypeptide and integrinligand is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction.Additionally, complex formation within reaction mixtures containing thetest compound and variant integrin polypeptide can also be compared tocomplex formation within reaction mixtures containing the test compoundand the corresponding wild-type integrin polypeptide.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the variant integrinpolypeptide or the binding partner (i.e., the integrin ligand) onto asolid phase, and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the variant integrin polypeptides and thebinding partner (e.g., an integrin ligand), e.g., , by competition, canbe identified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the variant integrin polypeptideor the integrin ligand, is anchored onto a solid surface (e.g., amicrotiter plate), while the non-anchored species is labeled, eitherdirectly or indirectly. The anchored species can be immobilized bynon-covalent or covalent attachments. Alternatively, an immobilizedantibody specific for the species to be anchored can be used to anchorthe species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the variant integrinpolypeptide and the integrin ligand is prepared in that either thevariant integrin polypeptide or the integrin ligand is labeled, but thesignal generated by the label is quenched due to complex formation (see,e.g., U.S. Pat. No. 4,109,496 that utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt variant integrin polypeptide-binding partnerinteraction can be identified.

In yet another aspect, the variant integrin polypeptide proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223–232; Madura et al. (1993) J. Biol. Chem. 268:12046–12054; Bartelet al. (1993) Biotechniques 14:920–924; Iwabuchi et al. (1993) Oncogene8:1693–1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with variant integrin polypeptide.

A two-hybrid assay can be carried out using a variant A domain as thebait protein. Briefly, the variant A domain is fused to the LexA DNAbinding domain and used as bait. The prey is an aptamer library clonedinto the active site loop of TrxA expression as a fusion protein with anN-terminal nuclear localization signal, a LexA activation domain, and anepitope tag (Colas et al. 1996 Nature 380:548; and Gyuris et al. Cell1993 75:791). Yeast cells are transformed with bait and prey genes. Ifan aptamer binds to variant A domain, the LexA activation domain isbrought into proximity with the LexA DNA binding domain and expressionof genes having an appropriately positioned LexA binding site increases.

To test this system, yeast strain EGY48 was transformed with the baitplasmid and pSH18–34 (a URA3, 2 μm plasmid containing the GAL1 promoterfused to lacZ, in which the GAL1 enhancer-like Upstream ActivatingSequence (UASG) has been replaced with binding sites for eight LexAdimers). The yeast strain contains two reporter genes, LexAop-LEU2(replaces the yeast chromosomal LEU2 gene) and LexAop-lacZ, carried on a2 μm HIS3⁺ plasmid). Transformed cells were plated on medium containingX-gal. The presence of the bait did not cause the expression ofβ-galactosidase. The bait by itself also did not activate theLexAop-Leu2 gene in EGY48, since the transformed cells did not grow onLeu deficient plates. Next, nuclear transport of the bait was assessedusing the bait plasmid and a pJK101 reporter in a repression type assay.In this assay DNA binding by transcriptionally inert LexA fusionproteins can be detected. The pJK101 reporter is a URA3, 2 μm plasmidcontaining most of the UASG. A high affinity binding site for lexA islocated between the Gal1 transcription start and the UASG. CD11bA-LexAbait impaired the galactosidase activity of yeast harboring the pJK101plasmid when grown on galactose medium, indicating that this bait entersthe nucleus.

An aptamer library was introduced into the Leu and lacZreporter-containing yeast cells. Synthesis of library proteins wasinduced by growing the yeast in galactose medium. In the absence of asuitable prey that binds the bait, the yeast cells does not grow on Leu⁻medium and have no β-galactosidase activity. A cell expressing asuitable prey will form colonies on Leu⁻ medium and have β-galactosidaseactivity. Selective galactose (but not glucose) inducible expressionallows the Leu and LacZ phenotypes to be unambiguously ascribed to thelibrary protein, diminishing the number of library plasmids that must beexcluded by subsequent analysis. Plasmids from positive colonies (i.e.,colonies able to grow on galactose, Ura⁻, Trp⁻ His⁻ Leu⁻ plates) wererescued (Hoffman et al. 1987 Gene 57:267). Before the respective cloneswere further characterized, the specificity of their interaction withthe bait was tested. This was done by showing that they do not interactwith unrelated or nonfunctional baits (e.g., a CD11a A-domain-lexA and11bA-D242A-lexA respectively), with the DNA-binding domain portion ofthe bait or nonspecifically with the promoters or other elements of thetranscription machinery. The library plasmids were rescued from thegalactose-dependent Leu+ lacZ+ yeast and re-introduced into the originalselection strain and into other strains containing different baits.Specific interactors confer the galactose-dependent Leu+ and lacZ+phenotype to yeast containing the original bait, but not to yeastcontaining unrelated baits. An interaction mating assay can be used totest specificity. Briefly, a strain that contains the prey is mated withdifferent yeast strains which express either the original bait proteinor the control bait proteins. Reporters should only be active indiploids that contain the original bait (Finley et al. (1994) Proc.Nat'l Acad. Sci USA 91:12980).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., an variant integrin polypeptide modulating agent) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

Pharmaceutical Compositions

The variant integrin polypeptides, fragments thereof, as well asanti-variant integrin polypeptide antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions. Such compositions typically include theprotein or antibody and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and anti-fungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, intravenous, intradermal, subcutaneous, oral,inhalation, transdermal, transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indeces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e,. including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin or a protein such asa cytokine or interleukin.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054–3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Animal Models of Processes Involving Integrins

A model of vascular injury that can be useful in testing potentialtherapeutic compositions is described by Simon et al. (J. Clin. Invest.2000 105:293). Tang et al. (1997 J Exp Med 186:1853) describe CD11bknockout mice which can be useful in various screening assays. An animalmodel of bum injury may also be useful (Plast Reconstr Surg 199596:1177).

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted integrinexpression or activity.

“Treatment” or “treating a patient”, as used herein, is defined as theapplication or administration of a therapeutic agent to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease, a symptom ofdisease or a predisposition toward a disease, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improveor affect the disease, the symptoms of disease or the predispositiontoward disease. A therapeutic agent includes, but is not limited to,small molecules, peptides, antibodies, ribozymes and antisenseoligonucleotides.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

1. A purified polypeptide consisting of amino acid 1–188 of SEQ ID NO:1.